sq-23377 and arachidonyltrifluoromethane

We observed that human (Jurkat) T-cells constitutively expressed the mRNA, encoding for the four isoforms of phospholipase A(2) (PLA(2)), i.e. two secretory (type IB and type V), and two cytosolic (type IV, Ca(2+)-dependent and type VI, Ca(2+)-independent). In order to assess whether these PLA(2) isoforms are active, we labeled Jurkat T-cells with [(3)H]arachidonic acid ([(3)H]AA) and determined its release into the extracellular medium in the presence of phorbol 12-myristate 13-acetate (PMA) and ionomycin. The three PLA(2) isoforms seem functional as aristolochic acid and bromoenol lactone (BEL), the respective inhibitors of type IB/type V and type VI PLA(2)s, significantly inhibited the release of free [(3)H]AA. On the other hand, arachidonyl trifluoromethyl ketone (AACOCF(3)), an inhibitor of type IV PLA(2), failed to curtail significantly the release of free [(3)H]AA into the extracellular medium. We assessed the implication of these PLA(2) isoforms in transcription of the interleukin-2 (IL-2) gene, involved in T-cell proliferation. Hence, aristolochic acid and BEL, but not AACOCF(3), significantly inhibited the PMA and ionomycin-induced induction of mRNA of IL-2. Similarly, aristolochic acid and BEL, but not AACOCF(3), significantly inhibited the PMA and ionomycin-induced secretion of IL-2 in the culture supernatants. Together these results suggest that human Jurkat T-cells possess two secretory and two cytosolic PLA(2) isoforms and only three of them (type IB, type V and type VI) are implicated in T-cell proliferation.

Topics: Arachidonic Acid; Arachidonic Acids; Calcium; Cell Division; Enzyme Inhibitors; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Humans; Interleukin-2; Ionomycin; Isoenzymes; Jurkat Cells; Phospholipases A; RNA, Messenger; Tetradecanoylphorbol Acetate; Tritium

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; Cannabinoids; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Ionomycin; Ionophores; Mice; Neuroblastoma; Neurons; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Subcellular Fractions; Tumor Cells, Cultured